The aim of this project has been to understand the molecular mechanisms that control the assembly and folding of the MHC class I molecules. The MHC Class I molecules serve as the critical cell surface structures that sample intracellular peptide fragments for recognition by antigen-specific, MHC-restricted T lymphocytes. In this project we have sought to understand in an in vitro system how the components of this three chain structure consisting of the MHC class I molecule, peptide, and the light chain, beta2-microglobulin follow a pathway from their biosynthesis as separate chains to a single cell surface molecule. By engineering single chain analogues of these molecules for expression both in bacteria and in tissue culture cells, we have been able to examine the contribution of the peptide and light chain components to the final structure. This has allowed us to develop a strategy for the production of cell lines that express only one class I MHC molecule and to generate transgenic animals that express single chain class I molecules. These studies should allow us not only to understanding basic rules of protein folding as they pertain to the trimer MHC class I molecule, but also allow us to exploit this knowledge in generating animal models with highly restricted MHC class I expression.